Identification of potential biomarkers in active Lyme borreliosis

Objectives Lyme serology does not readily discriminate an active Lyme borreliosis (LB) from a previous Borrelia infection or exposure. Here, we aimed to investigate a large number of immunological protein biomarkers to search for an immunological pattern typical for active LB, in contrast to patterns found in healthy blood donors, a proportion of whom were previously exposed to Borrelia. Methods Serum samples from well-characterised adult patients with ongoing LB and healthy blood donors were included and investigated using a proximity extension assay (provided by Olink®) by which 92 different immune response-related human protein biomarkers were analysed simultaneously. Results In total, 52 LB patients and 75 healthy blood donors were included. The blood donors represented both previously Borrelia exposed (n = 34) and not exposed (n = 41) based on anti-Borrelia antibody status. Ten of the examined 92 proteins differed between patients and blood donors and were chosen for further logistic regression (p<0.1). Six proteins were statistically significantly different between LB patients and blood donors (p<0.05). These six proteins were then combined in an index and analysed using receiver-operating-characteristic curve analysis showing an area under the curve of 0.964 (p<0.001). Conclusions The results from this study suggest that there is an immunological protein pattern that can distinguish a present Borrelia infection from a previous exposure as well as anti-Borrelia antibody negative blood donors. Although this method is not adapted for routine clinical use at this point, the possibility is interesting and may open new diagnostic opportunities improving the laboratory diagnostics of LB.

Objectives Lyme serology does not readily discriminate an ongoing Lyme borreliosis (LB) infection from a previous exposure. Here, we aimed to investigate a large number of immunological protein biomarkers to search for an immunological pattern typical for active LB, in contrast to patterns found in individuals previously exposed for Borrelia and healthy blood donors. Methods Serum samples from well-characterised adult patients with ongoing LB and healthy blood donors were included and investigated using a proximity extension assay (provided by Olink®) by which 92 different immune response-related human protein biomarkers were analysed simultaneously. Results In total, 52 LB patients and 75 healthy blood donors were included. The blood donors represented both previously Borrelia exposed (n=34) and not exposed (n=41) based on anti-Borrelia antibody status. Ten of the examined 92 immune-related proteins differed between patients and blood donors and were chosen for further logistic regression (p<0.1). Six proteins were statistically significant different between LB patients and blood donors (p<0.05); PPP1R9B, PRDX5, ITM2A, EIF4GI, DDX58, and ITGB6. These six proteins were then combined in an index and analysed using receiver-operating-characteristic curve analysis showing an area under the curve of 0.964 (p<0.001).

Conclusions
The results from this study suggest that there is an immunological protein pattern that can distinguish a present Borrelia infection from a previous exposure as well as anti-Borrelia antibody negative blood donors. Although this method is not adapted for routine clinical use at this point, the possibility is interesting and may open new diagnostic opportunities improving the laboratory diagnostics of LB. The study was approved by the respective regional ethical review boards in Sweden, Åland and Norway. For Sweden 2013/238-31, 2014/326-32, 2016/211-31, 2019 for Åland the approval was given at the regional ethical review board meeting no 2/2014 and for Norway 2014/1100 and additionally September 13, 2019 with reference no 9463. All study participants agreed to take part in the study and signed a written informed consent form.

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In total, 52 LB patients and 75 healthy blood donors were included. The blood donors 63 represented both previously Borrelia exposed (n=34) and not exposed (n=41)  The results from this study suggest that there is an immunological protein pattern that can    Table 1. 147 Parameters with at least 50% values within the detectable range were chosen for the 148 subsequent analysis. The further analyses were as follows: 1) The database was randomly 149 divided in two halves, equally for both LB patients and blood donors, and the difference 150 between the two groups within each half analysed separately with Mann-Whitney's U-test.

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This was done in order to reduce random statistical effects. 2) Secondly, the differences 152 between LB patients and blood donors, with a significance level of <0.1 simultaneously for 153 the two groups were qualified for successive analyses with multivariate logistic regression 154 with p<0.05 as a criterion. These were then subjected to form an index using a linear equation 155 with the log(OR) as weights for the various proteins, then presented in receiver-operating 156 characteristic (ROC) analysis. Statistical analyses were performed in Statistica version 13.

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The study was approved by the respective regional ethical review boards in Sweden, Åland

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Basic demographics of the LB patients and the blood donors are shown in Table 1. The LB 168 patients (n=52) consisted of the following manifestations; Lyme neuroborreliosis (n=41), 169 acrodermatitis chronica atrophicans and/or Lyme arthritis (n=7) and erythema migrans (n=4).

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Further details regarding the subjects may be found in the publication by Lager et al [12].  Table   178 1. In total, ten of the 92 variables showed a difference with p<0.1, further included in a 179 logistic regression model in which six (PPP1R9B, PRDX5, ITM2A, EIF4GI, DDX58, 180 ITGB6) of the ten protein markers remained (p<0.05), Table 2. Furthermore, an index was 181 created using the individual results for these six protein markers:   Table).  reported to play a significant role as a link between actin cytoskeleton and the plasma 214 membrane at synaptic junctions as well as a G-protein signalling regulator. It also appears that 215 this protein is of special importance in the nervous system [14,15]. PRDX5, peroxiredoxin-5, 216 also known as PMP20 is a thiol-specific peroxidase that catalyses reduction of organic

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Regarding limitations, although a reasonable number of LB patients and blood donors were 253 investigated, the material is still limited in number, and follow-up samples were not available 254 for the patients which would have been a valuable source of confirmation. Also, the number 255 of studied parameters in relation to the number of available samples needs to be handled with 256 caution in order to avoid random statistical effects.

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To conclude, we here show a possibility to discriminate ongoing LB from previous exposure